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Autophagy, a regulated nutrient recycling program can affect both cell survival and cell death. Here, we show that Ormeloxifene (ORM), a selective estrogen receptor modulator approved for oral contraceptive use induces autophagic flux in ovarian cancer cells, which is activated by an ER stress response upstream of autophagy. The ER stress response is characterized by activation of IRE1α, PERK and ATF6 and is under regulation of JNK. Pharmacological inhibition of either autophagy or ER stress increased cell survival, as did silencing of autophagy proteins LC3 and Beclin 1, implying that ORM-induced autophagy is pro-death in nature. Ultrastructural observations of treated cells confirmed stages of autophagic maturation. Caspase-dependent apoptosis succeeded these events and was characterized by generation of reactive oxygen species and disruption of mitochondrial membrane potential. A concomitant inhibition of the Akt/mTOR axis was also observed with possible regulation of Akt by ORM. ORM inhibited tumor growth in ovarian xenograft model and displayed autophagic activity. In summary, in vitro and in vivo results reveal that ORM induces autophagy-associated cell death to attenuate proliferation of ovarian cancer cells. Our results demonstrate that using ORM in combination with ER stress and autophagy modulators could offer better therapeutic outcome in ovarian cancer.

Purpose: Mycobacterium tuberculosis (Mtb) inhibits host defense mechanisms, including autophagy. We investigated particles containing rapamycin (RAP) alone or in combination with isoniazid (INH) and rifabutin (RFB) for: targeting lung macrophages on inhalation; inducing autophagy; and killing macrophage-resident Mtb and/or augmenting anti-tuberculosis (TB) drugs., Methods: PLGA and drugs were spray-dried. Pharmacokinetics, partial biodistribution (LC-MS/MS) and efficacy (colony forming units, qPCR, acid fast staining, histopathology) in mice following dry powder inhalation were evaluated., Results: Aerodynamic diameters of formulations were 0.7-4.7 μm. Inhaled particles reached deep lungs and were phagocytosed by alveolar macrophages, yielding AUC0-48 of 102 compared to 0.1 μg/ml × h obtained with equivalent intravenous dose. RAP particles induced more autophagy in Mtb-infected macrophages than solutions. Inhaled particles containing RAP alone in daily, alternate-day and weekly dosing regimens reduced bacterial burden in lungs and spleens, inducing autophagy and phagosome-lysosome fusion. Inhalation of particles containing RAP with INH and RFB cleared the lungs and spleens of culturable bacteria., Conclusions: Targeting a potent autophagy-inducing agent to airway and lung macrophages alone is feasible, but not sufficient to eliminate Mtb. Combination of macrophage-targeted inhaled RAP with classical anti-TB drugs contributes to restoring tissue architecture and killing Mtb.

We investigated whether particles suitable for delivery to alveolar macrophages may provide a means of targeting rapamycin, an inducer of autophagy, to alveolar macrophages as a host-directed antituberculosis agent. Inhalable particles were prepared by spray-drying and characterized using laser scattering and electron microscopy. Their aerodynamic diameter was calculated from bulk and tapped densities. In vitro drug release was studied in PBS containing 1% SDS. In vitro uptake of particles by THP-1 derived macrophages was studied by flow cytometry. Cytotoxicity of the particles toward macrophages and their efficacy against intracellular Mycobacterium tuberculosis were studied using a methyltetrazolium assay and counting bacterial colonies obtained when cell lysates were plated on agar. The encapsulation efficiency was 88.8 ± 1.13% and drug content 22 ± 4% w/w. The particles had a median diameter of 2.88 ± 0.8 μm and appeared as collapsed spheres. Their calculated aerodynamic diameter was about 1 μm. In vitro drug release from the particles was first-order and extended beyond 10 days. Flow cytometry indicated that the particles were taken up by macrophages within 3 h. Macrophages exposed to the particles or rapamycin in solution at a concentration of 100 μg/mL over a 24 h period maintained 79.37 ± 0.72% and 58.33 ± 1.39% viability, respectively. Efficacy studies concluded that particles were more effective in clearing intracellular mycobacteria than rapamycin in solution. It was concluded that the preparation was suitable for formulating as a dry powder inhalation to test efficacy of inhaled, macrophage-targeted rapamycin against TB.

Estrogen receptor negative (ER(-ve)) and p53 mutant breast tumors are highly aggressive and have fewer treatment options. Previously, we showed that molecular Iodine (I(2)) induces apoptosis in hormone responsive MCF-7 breast cancer cells, and non-apoptotic cell death in ER(-ve)-p53 mutant MDA-MB231 cells (Shrivastava, 2006). Here we show that I(2) (3 μM) treatment enhanced the features of autophagy in MDA-MB231 cells. Since autophagy is a cell survival response to most anti-cancer therapies, we used both in vitro and in vivo systems to determine whether ER(-ve) mammary tumors could be sensitized to I(2)-induced apoptosis by inhibiting autophagy. Autophagy inhibition with chloroquine (CQ) and inhibitors for PI3K (3MA, LY294002) and H+/ATPase (baflomycin) resulted in enhanced cell death in I(2) treated MDA-MB231 cells. Further, CQ (20 μM) in combination with I(2), showed apoptotic features such as increased sub-G1 fraction (∼5-fold), expression of cleaved caspase-9 and -3 compared to I(2) treatment alone. Flowcytometry of I(2) and CQ co-treated cells revealed increase in mitochondrial membrane permeability (p<0.01) and translocation of cathepsin D activity to cytosol relative to I(2) treatment. For in vivo studies ICRC mice were transplanted subcutaneously with MMTV-induced mammary tumors. A significant reduction in tumor volumes, as measured by MRI, was found in I(2) and CQ co-treated mice relative to I(2) or vehicle treated mice. These data indicate that inhibition of autophagy renders ER(-ve) breast tumor cells more sensitive to I(2) induced apoptosis. Thus, I(2) together with autophagy inhibitor could have a potential tumorostatic role in ER(-ve) aggressive breast tumors that may be evaluated in future studies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objectives: Given that autophagy inhibition is a feasible way to enhance sensitivity of cancer cells towards chemotherapeutic agents, identifying potent autophagy inhibitor has obvious clinical relevance. Here, we investigated ability of TN‐16, a microtubule disrupting agent, on modulation of autophagic flux and its significance in promoting in vitro and in vivo cancer cell death. Materials and methods: The effect of TN‐16 on cancer cell proliferation, cell division, autophagic process and apoptotic signalling was assessed by various biochemical (Western blot and SRB assay), morphological (TEM, SEM, confocal microscopy) and flowcytometric assays. In vivo anti‐tumour efficacy of TN‐16 was investigated in syngeneic mouse model of breast cancer. Results: TN‐16 inhibited cancer cell proliferation by impairing late‐stage autophagy and induction of apoptosis. Inhibition of autophagic flux was demonstrated by accumulation of autophagy‐specific substrate p62 and lack of additional LC3‐II turnover in the presence of lysosomotropic agent. The effect was validated by confocal micrographs showing diminished autophagosome‐lysosome fusion. Further studies revealed that TN‐16–mediated inhibition of autophagic flux promotes apoptotic cell death. Consistent with in vitro data, results of our in vivo study revealed that TN‐16–mediated tumour growth suppression is associated with blockade of autophagic flux and enhanced apoptosis. Conclusions: Our data signify that TN‐16 is a potent autophagy flux inhibitor and might be suitable for (pre‐) clinical use as standard inhibitor of autophagy with anti‐cancer activity. [ABSTRACT FROM AUTHOR]

7 Histopathology of lung in animals that were: (1) Infected, untreated (II) Infected + treated with Inhalation - Rap particles: daily dosing (I-RAP 10 D) for 10 days (III) Infected + Inhalation - Rap particles: alternate day dosing (I-RAP 10 ALT) (IV) Infected + Inhalation-Rap particles: weekly dosing (I-RAP 10 WKLY) (V) Infected + Inhalation - Rap trehalose particles particles: daily dosing (I-RAP 10 TRE-D) (VI) Infected + Oral Isoniazid-Rifabutin (125 µg) + Inhalation-Isoniazid Rifabutin particles: daily dosing (O-INH-RFB + I-INH-RFB-D) (VII) Infected + Oral Isoniazid-Rifabutin (125 µg) + Inhalation - Isoniazid Rifabutin particles: daily dosing (O-INH-RFB + I-RAP-D) (VIII) Infected + Inhalation - Isoniazid-Rifabutin-Rap particles: daily dosing (I-INH + RFB + RAP-D) (IX) Infected + Inhalation - Blank PLGA particles: daily dosing (I-BLK-D) Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. The image in Panel VI (earlier, "RAP-TRE") has been replaced with the correct image. [Extracted from the article]

Towards identification of novel therapeutic candidates, a series of quinazolinone-based acetamide derivatives were synthesized and assessed for their anti-leishmanial efficacy. Amongst synthesized derivatives, compounds F12, F27 and F30 demonstrated remarkable activity towards intracellular L. donovani amastigotes in vitro , with IC 50 values of 5.76 ± 0.84 μM, 3.39 ± 0.85 μM and 8.26 ± 1.23 μM against promastigotes, and 6.02 μM ± 0.52, 3.55 ± 0.22 μM and 6.23 ± 0.13 μM against amastigotes, respectively. Oral administration of compounds F12 and F27 entailed >85% reduction in organ parasite burden in L. donovani -infected BALB/c mice and hamsters, by promoting host-protective Th1 cytokine response. In host J774 macrophages, mechanistic studies revealed inhibition of PI3K/Akt/CREB axis, resulting in a decrease of IL-10 versus IL-12 release upon F27 treatment. In silico docking studies conducted with lead compound, F27 demonstrated plausible inhibition of Leishmania prolyl-tRNA synthetase, which was validated via detection of decreased proline levels in parasites and induction of amino acid starvation, leading to G 1 cell cycle arrest and autophagy-mediated programmed cell death of L. donovani promastigotes. Structure−activity analysis and study of pharmacokinetic and physicochemical parameters suggest oral availability and underscore F27 as a promising lead for anti-leishmanial drug development. [Display omitted] • A series of quinazolinone-based acetamide derivatives (F1 - F33) were synthesized. • Two compounds, F12 and F27 exhibited maximum in vitro anti-leishmanial activity. • Compounds F12 and F27 significantly reduced parasite burden in L. d -infected animals. • Compound F27 inhibited host PI3K/Akt/CREB axis-mediated IL-10 secretion. • Compound F27 induced autophagy-mediated apoptosis in L. donovani promastigotes. [ABSTRACT FROM AUTHOR]

Autophagy is considered as an important cell death mechanism that closely interacts with other common cell death programs like apoptosis. Critical role of autophagy in cell death makes it a promising, yet challenging therapeutic target for cancer. We identified a series of 1,2,3-triazole analogs having significant breast cancer inhibition property. Therefore, we attempted to study whether autophagy and apoptosis were involved in the process of cancer cell inhibition. The lead molecule, 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol (T-12) induced significant cell cycle arrest, mitochondrial membrane depolarization, apoptosis and autophagy in MCF-7 and MDA-MB-231 cells. T-12 increased reactive oxygen species and its inhibition by N-acetyl- l -cysteine protected breast cancer cells from autophagy and apoptosis. Autophagy inhibitor, 3-methyladenine abolished T-12 induced apoptosis, mitochondrial membrane depolarization and reactive oxygen species generation. This suggested that T-12 induced autophagy facilitated cell death rather than cell survival. Pan-caspase inhibition did not abrogate T-12 induced autophagy, suggesting that autophagy precedes apoptosis. In addition, T-12 inhibited cell survival pathway signaling proteins, Akt, mTOR and Erk1/2. T-12 also induced significant regression of tumor with oral dose of as low as 10 mg/kg bodyweight in rat mammary tumor model without any apparent toxicity. In presence of reactive oxygen species inhibitor (N-acetyl- l -cysteine) and autophagy inhibitor (chloroquine), T-12 induced tumor regression was significantly decreased. In conclusion, T-12 is a potent inducer of autophagy-dependent apoptosis in breast cancer cells both in vitro and in vivo and can serve as an important lead in development of new anti-tumor therapy. [ABSTRACT FROM AUTHOR]

Therapy resistance by evasion of apoptosis is one of the hallmarks of human cancer. Therefore, restoration of cell death by non-apoptotic mechanisms is critical to successfully overcome therapy resistance in cancer. By rational drug design approach, here we try to provide evidence that subtle changes in the chemical structure of spisulosine completely switched its cytotoxic function from apoptosis to autophagy. Our most potent molecule (26b) in a series of 16 synthesized derivatives showed robust autophagic cell death in diverse cancer cells sparing normal counterpart. Compound 26b mediated lethal autophagy induction was confirmed by formation of characteristic autophagic vacuoles, LC3 puncta formation, upregulation of signature autophagy markers like Beclin and Atg family proteins. Altogether, we have detected novel autophagy inducer small molecule which can be tested further for drug discovery research. Compound 26b promotes Autophagic cell death. Image 1 • New Spisulosine Derivative as novel autophagy inducer. • Robust autophagic cell death in diverse cancer cells. • Sparing normal counterpart. [ABSTRACT FROM AUTHOR]